Proper localization of proteins is crucial for all cells. The signal recognition particle (SRP) and its receptor (SR) constitute the major cellular machinery that mediates the co-translational targeting of roughly one third of cellular proteins to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. Although rapid progress has been made in understanding the SRP pathway, many fundamental aspects of its molecular mechanism remain to be elucidated. Our general goal is to decipher, at a biochemical and biophysical level, the intricate inner workings of this universally conserved targeting machine. Our specific goal is to decipher the molecular mechanisms by which the loading of the cargo proteins on the SRP is coupled to its rapid delivery and efficient unloading to the translocation machinery, and to begin understand the mechanism of SRP in the context of the crowded environment at the ribosome exit site. To this end, three aims are envisioned: (1) We will decipher the role of the SRP RNA in mediating the communication between the cargo and the SRP and SR GTPases; (2) We will elucidate the precise mechanisms by which the cargo protein is transferred from the targeting to the translocation machinery; and (3) We will decipher whether and how the major co-translational chaperone, trigger factor, modulates the efficiency and fidelity of the SRP pathway. Ultimately, these studies will not only advance our understanding of the process of protein localization within the cell, but also provide new insights into the general principles of molecular recognition and regulation at a fundamental level. The proposed research is of a most basic nature, and will contribute profoundly to our general understanding of physiology and pathology of all living cells at the molecular level. PUBLIC HEALTH RELEVANCE: Proper localization of proteins is essential for all cells. Mislocalization of proteins cause impaired cell growth, function, and eventually cell death, leading to devastating diseases such as cystic fibrosis, diabetes, and neurodegeneration. The proposed studies will significantly advance our understanding of the mechanism of membrane protein localization and biogenesis within the cell, and contribute profoundly to our general understanding of physiology and pathology of all living cells at a molecular level.